Investigation of non-reversible and reversible ezymatic reactions

Tirtas grįžtamos ir negrįžtamos fermentinių reakcijų produktų kaupimasis, kuris buvo modeliuojas ir tiriamas eksperimentiškai. Eksperimentiniai duomenys buvo gauti naudojant šarminę fosfatazę (EC 3.1.3.1) ir ADH (EC 1.1.1.1). Modeliuojant negrįžtamą reakciją produkto kaupimasis gali būti tiesiškas (kai substrato koncentracija yra pakankamai didelė), tuo tarpu grįžtama reakcija nėra tiesiška. Tai buvo patvirtinta eksperimentiškai. Nustatyta, kad fermentų apsukos yra efosfatazė=0,5 s–1, eADH=4,8 s–1 ir fermento-substrato kvazi-pusiausvirinės disociacijos konstantos yra ξfosfatazė=0,1 M, ξADH=0,6 M.Time-course of product accumulation of both non-reversible and reversible enzymatic reactions was modeled and investigated experimentally. For the experiments alkaline phosphatase (EC 3.1.3.1) and ADH (EC 1.1.1.1) were used. It follows from the modeling that product accumulation may be close to linear (if substrate concentration is sufficiently high) in the case of non-reversible reaction, while in that of reversible is essentially nonlinear. That is confirmed experimentally. It has been found that the enzyme turnover is ephosphotase =0,5 s–1, eADH =4,8 s–1, the substrate–enzyme quasi-equilibrium dissociation constant being phosphotase = 0,1 M, ADH = 0,6 M

Engineering Approach to Enzyme Activity

Konkretų fermentą galima apibrėžti dviem parametrais – katalitine konstanta, apibūdinančia efektyvumą ir fermento-substrato komplekso disociacijos konstanta, nusakančia specifiškumą substratui. Tradiciniai šių parametrų nustatymo būdai susiję su liestinės brėžimu per pradinius taškus kinetinėje kreivėje ir koordinačių transformacijomis. Per pradinius taškus brėžiant liestinę laikoma, kad fermentui idealios sąlygos yra tik proceso pradžioje, bet taip apskaičiuota reikšmė neatspindi realios situacijos, nes apibūdina tik pirmąsias reakcijos sekundes ir daroma prielaida, kad reakcijos greitis nekinta. Tuo tarpu matematinis modeliavimas suteikia galimybę panaudoti ne tik pirmuosius, bet ir vėliau matuotus duomenis, bei apsieiti be koordinačių transformacijos, todėl yra geresnis ir patogesnis už tradicinius metodus. Apskaičiuoti parametrai turi ir praktinį pritaikymą, kadangi leidžia numatyti, kokie komponentų kiekiai bus sistemoje tam tikru laiko momentu. Eksperimentiškai tirta šarminės fosfatazės (ALP) ir alkoholdehidrogenazės (ADH) kinetika bei atitinkamai sukurti matematiniai modeliai negrįžtamai ir grįžtamai katalizei.The activity of an enzyme depends solely on its efficacy and the affinity of the substrate for the enzyme. The enzyme, therefore, usually can be characterized by two parameters, the catalytic constant and equilibrium dissociation constant. Initial rate of enzymatic reaction is usually determined from the tangent of product accumulation curve using only a small part of experimental data. Determining the dissociation constant usually involves coordinate transformation impeding the interpretation of results and often leading to uncontrollable errors. Such methods are not justified now when computers and computer programs are easily accessible. Mathematical mo-deling using the computers both helps understanding the processes and enables to estimate the parameters more accurately. In this investigation both the modeling and experiments were performed. Alkaline phosphatase (ALP) and alcohol dehydrogenase (ADH) were used for the investigation. Keywords: ADH, ALP, catalysis, enzyme, Henri, mathematical modeling, product, substrate

Sodium Is Not Required for Chloride Efflux via Chloride/Bicarbonate Exchanger from Rat Thymic Lymphocytes

Sodium-dependent Cl−/HCO3- exchanger acts as a chloride (Cl−) efflux in lymphocytes. Its functional characterization had been described when Cl− efflux was measured upon substituting extracellular sodium (Na+) by N-methyl-D-glucamine (NMDG). For Na+ and Cl− substitution, we have used D-mannitol or NMDG. Thymocytes of male Wistar rats aged 7–9 weeks were used and intracellular Cl− was measured by spectrofluorimetry using MQAE dye in bicarbonate buffers. Chloride efflux was measured in a Cl−-free buffer (Cl− substituted with isethionate acid) and in Na+ and Cl−-free buffer with D-mannitol or with NMDG. The data have shown that Cl− efflux is mediated in the absence of Na+ in a solution containing D-mannitol and is inhibited by H2DIDS. Mathematical modelling has shown that Cl− efflux mathematical model parameters (relative membrane permeability, relative rate of exchanger transition, and exchanger efficacy) were the same in control and in the medium in which Na+ had been substituted by D-mannitol. The net Cl− efflux was completely blocked in the NMDG buffer. The same blockage of Cl− efflux was caused by H2DIDS. The study results allow concluding that Na+ is not required for Cl− efflux via Cl−/HCO3- exchanger. NMDG in buffers cannot be used for substituting Na+ because NMDG inhibits the exchanger

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